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Introduction: One of the common causes of 46,XY differences in sex development (DSD) cases is androgen insensitivity syndrome. This X-linked recessive inherited condition is associated with pathological variations of the AR gene, leading to defects in androgen action. Affected 46,XY infants or individuals experience variable degrees of undervirilization and those with severe form will have female-like external genitalia. Therefore, they were more likely assigned and reared as females. The confirmatory molecular test is often needed due to similar clinical manifestations with other conditions causing 46,XY DSD. Since in our country, the molecular test for the AR gene is lacking, the study is conducted as a preliminary study to elaborate on the possibility of developing a molecular test for the AR gene in 46,XY DSD cases. Methods: Archived DNAs of 13 46,XY DSD cases were analyzed using polymerase chain reaction and direct sequencing for molecular defects in the AR gene. Clinical and hormonal data were collected and analyzed. Results: The study successfully amplified and visualized the eight exons of the AR gene and revealed two subjects carrying AR gene variants at exon 7. In the first case, 1.2-year-old boy carried heterozygous p.Gln825Arg, which has never been reported elsewhere, and the second subject, a 2.1-year-old girl with heterozygous p.Arg841His. Both subjects presented with severe undervirilization of external genitalia with external genitalia masculinization scores (EMS) of 1.5 and 3. Conclusion: In this series, two of 13 46,XY DSD cases carried variants at the AR gene, resulting in complete androgen insensitivity syndrome. [ABSTRACT FROM AUTHOR]

Aim: This study examines the distribution of genital abnormalities based on physical examination of our patients with 46, XY disorders of sex development (46, XY DSD), and aims to define severity and frequency of hypospadias in 46, XY DSD. Hypospadias is a relatively prevalent congenital anomaly. Although genetic, environmental and hormonal factors are considered to be responsible, etiology is not clarified in several hypospadias cases. Materials and Methods: Clinical, laboratory and genetic records of all cases with 46, XY DSD, who were evaluated by the sex determination monitoring board were retrospectively reviewed. In the diagnosis, hypospadias cases were examined in terms of the place of hypospadias and coexisting other external genital findings. Results: There were 72 patients with 46, XY DSD. 5-α reductase deficiency [n=32 (44.4%)] was the most commonly encountered diagnosis followed by androgen insensitivity syndrome [n=26, (36.1%)]. Proximal hypospadias were presented in 44.4% (n: 32) of the cases and only 6 of them (18.8%) were isolated hypospadias. In 81.2% of these cases, at least one of the anomalies such as cordi, bifid scrotum, undescended testis and micropenis accompanied proximal hypospadias. None of the distal hypospadias cases were referral clinic finding. Conclusions: 46 XY DSD is a heterogeneous group of patients with a varying age of presentation and a diverse clinical profile. It can be stated that proximal hypospadias is the most common referral clinic finding of 46, XY DSD, and the risk of 46, XY DSD increases with the intensifying degree of hypospadias and the presence of coexisting genital abnormalities such as cordi, bifid scrotum, undescended testis and micropenis. [ABSTRACT FROM AUTHOR]

Missense variants in the RNA-helicase DHX37 are associated with either 46,XY gonadal dysgenesis or 46,XY testicular regression syndrome (TRS). DHX37 is required for ribosome biogenesis, and this subgroup of XY DSD is a new human ribosomopathy. In a cohort of 140 individuals with 46,XY DSD, we identified 7 children with either 46,XY complete gonadal dysgenesis or 46,XY TRS carrying rare or novel DHX37 variants. A novel p.R390H variant within the RecA1 domain was identified in a girl with complete gonadal dysgenesis. A paternally inherited p.R487H variant, previously associated with a recessive congenital developmental syndrome, was carried by a boy with a syndromic form of 46,XY DSD. His phenotype may be explained in part by a novel homozygous loss-of-function variant in the NGLY1 gene, which causes a congenital disorder of deglycosylation. Remarkably, a homozygous p.T477H variant was identified in a boy with TRS. His fertile father had unilateral testicular regression with typical male genital development. This expands the DSD phenotypes associated with DHX37. Structural analysis of all variants predicted deleterious effects on helicase function. Similar to all other known ribosomopathies, the mechanism of pathogenesis is unknown. [ABSTRACT FROM AUTHOR]

Keywords: gonadal dysgenesis; haematological chimerism; sex-discordant; twins; XY DSD EN gonadal dysgenesis haematological chimerism sex-discordant twins XY DSD 487 489 3 04/30/20 20200501 NES 200501 46,XY Disorders of sex differentiation (DSD) such as complete gonadal dysgenesis, disorders of androgen synthesis and action, ovotesticular DSD can present with unambiguous or minimally virilized female external genitalia. Genotype analysis on lymphocyte DNA from proband's twin brother showed the presence of a female genotype, which was identical to proband's genotype. Genotype analysis and karyotyping of the proband's twin brother's lymphocytes showed a genotype profile similar to the proband and a 46,XY [98%]/46,XX [2%] karyotype. [Extracted from the article]

Empty follicle syndrome (EFS) is a disorder associated with female infertility and presents as a complete failure to retrieve oocytes during ART cycles despite normal follicle development and careful aspiration. To date, only two EFS cases have been reported with homozygous missense mutations in the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) gene, and both cases showed normal estradiol (E2) production during ovulation induction. The molecular genetic mechanisms of EFS remain unknown. Herein, we report two novel homozygous inactivating LHCGR mutations, c.736 C>T (p.Q246*) and c.846dupT (p.R283*), in two female EFS patients from unrelated consanguineous families. The probands had impaired E2 production during the ART process, which differs from previously reported EFS cases. The inactivating mutations not only led to EFS in the two female probands, but also resulted in 46, XY disorder of sex development (46, XY DSD) in their male siblings. As far as we know, this is the first report of LHCGR mutations leading to both EFS and 46, XY DSD within the same pedigree. Our findings provide researchers and clinicians with a better understanding of phenotype-genotype correlations between EFS and 46, XY DSD and the LHCGR gene. [ABSTRACT FROM AUTHOR]

Background: Disorders of sex development (DSD) have an estimated frequency of 0.5% of live births encompassing a variety of urogenital anomalies ranging from mild hypospadias to a discrepancy between sex chromosomes and external genitalia. In order to identify the underlying genetic etiology, we had performed exome sequencing in a subset of DSD cases with 46,XY karyotype and were able to identify the causative genetic variant in 35% of cases. While the genetic etiology was not ascertained in more than half of the cases, a large number of variants of unknown clinical significance (VUS) were identified in those exomes. Methods: To investigate the relevance of these VUS in regards to the patient's phenotype, we utilized a mouse model in which the presence of a Y chromosome from the poschiavinus strain (YPOS) on a C57BL/6J (B6) background results in XY undervirilization and sex reversal, a phenotype characteristic to a large subset of human 46,XY DSD cases. We assessed gene expression differences between B6-YB6 and undervirilized B6-YPOS gonads at E11.5 and identified 515 differentially expressed genes (308 underexpressed and 207 overexpressed in B6-YPOS males). Results: We identified 15 novel candidate genes potentially involved in 46,XY DSD pathogenesis by filtering the list of human VUS-carrying genes provided by exome sequencing with the list of differentially expressed genes from B6-YPOS mouse model. Additionally, we identified that 7 of the 15 candidate genes were significantly underexpressed in the XY gonads of mice with suppressed Sox9 expression in Sertoli cells suggesting that some of the candidate genes may be downstream of a well-known sex determining gene, Sox9. Conclusion: The use of a DSD-specific animal model improves variant interpretation by correlating human sequence variants with transcriptome variation. [ABSTRACT FROM AUTHOR]

Objective Disorders of sex development ( DSD) are a heterogeneous group of conditions affecting the differentiation and development of the internal and external genitalia. Here, we aimed at identifying the genetic cause of DSD in two 46, XY sisters from a consanguineous family. Design We performed a whole-exome sequencing of two 46, XY female individuals. Sanger sequencing was used to validate the most likely candidate variant, affecting the desert hedgehog ( DHH) gene. Molecular dynamics simulations were performed to get insights into the impact of the variant on protein structure and on its interaction with the protein partner BOC (brother of CDO/cell adhesion molecule, downregulated by oncogenes). Patients The index patient presented with a female phenotype, primary amenorrhoea (low oestradiol and testosterone and high FSH and LH). She also had an apparent absence of intra-abdominal gonads and uterus, facial dysmorphy, psychomotor retardation and neuropathy. Her sister displayed a similar gonadal and endocrinological picture, without dysmorphy or psychomotor retardation. Results Whole-exome sequencing revealed a homozygous variant in DHH leading to the p.Trp173Cys substitution. The relevant Trp residue is conserved, and its alteration was predicted to be deleterious. Molecular dynamics simulations showed that the mutation increases the conformational flexibility of the protein and potentially alters its interaction with BOC, a positive regulator of Hedgehog signalling. We do not exclude an interference of the mutation with DHH-intein-mediated auto-processing. Conclusions This report increases the number of described homozygous DHH variants and highlights the importance of advanced bioinformatic tools to better understand the pathogenicity of human variants. [ABSTRACT FROM AUTHOR]

Study Question: What is the likelihood of identifying genetic or endocrine abnormalities in a group of boys with 46, XY who present to a specialist clinic with a suspected disorder of sex development (DSD)?Summary Answer: An endocrine abnormality of the gonadal axis may be present in a quarter of cases and copy number variants (CNVs) or single gene variants may be present in about half of the cases.What Is Known Already: Evaluation of 46, XY DSD requires a combination of endocrine and genetic tests but the prevalence of these abnormalities in a sufficiently large group of boys presenting to one specialist multidisciplinary service is unclear.Study, Design, Size, Duration: This study was a retrospective review of investigations performed on 122 boys.Participants/materials, Setting, Methods: All boys who attended the Glasgow DSD clinic, between 2010 and 2015 were included in the study. The median external masculinization score (EMS) of this group was 9 (range 1-11). Details of phenotype, endocrine and genetic investigations were obtained from case records.Main Results and the Role Of Chance: An endocrine abnormality of gonadal function was present in 28 (23%) with a median EMS of 8.3 (1-10.5) whilst the median EMS of boys with normal endocrine investigations was 9 (1.5-11) (P = 0.03). Endocrine abnormalities included a disorder of gonadal development in 19 (16%), LH deficiency in 5 (4%) and a disorder of androgen synthesis in 4 (3%) boys. Of 43 cases who had array-comparative genomic hybridization (array-CGH), CNVs were reported in 13 (30%) with a median EMS of 8.5 (1.5-11). Candidate gene analysis using a limited seven-gene panel in 64 boys identified variants in 9 (14%) with a median EMS of 8 (1-9). Of the 21 boys with a genetic abnormality, 11 (52%) had normal endocrine investigations.Limitations, Reasons For Caution: A selection bias for performing array-CGH in cases with multiple congenital malformations may have led to a high yield of CNVs. It is also possible that the yield of single gene variants may have been higher than reported if the investigators had used a more extended gene panel.Wider Implications Of the Findings: The lack of a clear association between the extent of under-masculinization and presence of endocrine and genetic abnormalities suggests a role for parallel endocrine and genetic investigations in cases of suspected XY DSD.Study Funding/competing Interest(s): RN was supported by the James Paterson Bursary and the Glasgow Children's Hospital Charity Summer Scholarship. SFA, RM and EST are supported by a Scottish Executive Health Department grant 74250/1 for the Scottish Genomes Partnership. EST is also supported by MRC/EPSRC Molecular Pathology Node and Wellcome Trust ISSF funding. There are no conflicts of interest.Trial Registration Number: None. [ABSTRACT FROM AUTHOR]

The 46, XY disorder of sex development (DSD) is the main cause of birth defects; however, as it is a group of highly heterogeneous diseases, >50% of cases are not accurately diagnosed. Identification of more cases will improve understanding of the relationship between genotype and phenotype for DSD. The present study conducted a systematic analysis of the clinical characteristics of a proband with 46, XY DSD, applied genetic analysis by whole-exome sequencing to this pedigree and performed bioinformatics analysis of the identified variant. The proband presented with a short penis, lack of testicles and partial growth hormone (GH) deficiency at 1 year old. Histopathological examination revealed there were oviduct, epididymis and fibrous vascular tissue on both sides of the abdomen. The last follow-up at 5 years of age revealed that the patient exhibited restricted growth, a 1.5-cm penis and lack of testicles. Notably, a novel pathogenic mitogen-activated protein kinase kinase kinase 1 (MAP3K1) variant (c.3020A>G) was identified in the proband, resulting in a change in the 1,007th amino acid (glutamine) of the encoded protein. This variant caused the uncharged neutral glutamine to be replaced by a positively charged basic arginine. p.Gln1007 in MAP3K1 was confirmed to be conserved across various species. Pathogenicity analysis using bioinformatics tools suggested that this MAP3K1 variant may cause functional defects. In conclusion, the present study identified a novel MAP3K1 variant that was the cause of 46, XY DSD and partial GH deficiency. The present findings extend the mutation spectrum of MAP3K1 and provide novel characteristics of 46, XY DSD. [ABSTRACT FROM AUTHOR]

Inactivating mutations of the 5 α-steroid reductase type-2 ( SRD5A2) gene result in a broad spectrum of masculinization defects, ranging from a male phenotype with hypospadias to a female phenotype with Wolffian structures. Molecular studies of the SRD5A2 revealed a new heterozygous gene variant within the coding region that results in phenotypic expression. A c.92C>T transition changing serine to phenylalanine at codon 31 of exon 1 (p.Ser31Phe) was identified in a patient with 46,XY disorder of sexual development who displayed glandular hypospadias with micropenis and bilateral cryptorchidism. The restoration of the p.Ser31Phe mutation by site-directed mutagenesis and transient expression assays using cultured HEK-293 cells showed that this novel substitution does not abolish but does deregulate the catalytic efficiency of the enzyme. Thus, the maximum velocity ( Vmax) value was higher for the mutant enzyme (22.5 ± 6.9 nmol DHT mg protein−1 h−1) than for the wild-type enzyme (9.8 ± 2.0 nmol DHT mg protein−1 h−1). Increased in vitro activity of the p.Ser31Phe mutant suggested an activating effect. This case provides evidence that heterozygous missense mutations in SRD5A2 may induce the abnormal development of male external genitalia. [ABSTRACT FROM AUTHOR]

At least one genetic defect in each reaction of the classical androgen biosynthesis pathway has been described. For some steps, such as the conversion of cholesterol to pregnenolone and the 17,20-lyase reaction, two or three genetic defects cause similar disorders with overlapping phenotypes and biochemical profiles. The elucidation of the molecular basis for these diseases has helped to define the pathways, essential genes, and enzymatic steps required to make androgens, and this knowledge is being exploited to develop better treatments of androgen-dependent diseases. Furthermore the description of nonclassical lipoid CAH and the protean manifestations of P450 oxidoreductase (POR) deficiencies has expanded the spectrum of human disease caused by disordered steroidogenesis. Finally, the recognition of the backdoor pathway to DHT has added a new dimension to our understanding of how steroid flux is maintained in normal and pathologic states. The traditional view of male external genital development has been that fetal testicular testosterone is converted to DHT by 5α-reductase Type 2 in genital skin, which then acts in a paracrine fashion to stimulate fusion of the labio-scrotal folds and phallic growth. This view is consistent with the incomplete external genital development in persons with severe deficiencies of 5α-reductase type 2. The new observations concerning AKR1C2/4 and the backdoor pathway indicate that DHT produced in the testis via the backdoor pathway also acts as a hormone to induce labio-scrotal fusion. Thus, both the classic and backdoor pathways are needed, and DHT acts in male genital development as both a paracrine factor and as a hormone. These surprising findings are revising our understanding of the mechanisms by which male sexual differentiation occurs, and illustrate the importance of detailed studies of rare patients with 46,XY DSD. [ABSTRACT FROM AUTHOR]

MAMLD1 (mastermind-like domain containing 1) is a recently discovered causative gene for 46,XY disorders of sex development (DSD), with hypospadias as the salient clinical phenotype. To date, microdeletions involving MAMLD1 have been identified in six patients, and definitive mutations (nonsense and frameshift mutations that are predicted to undergo nonsense mediated mRNA decay [NMD]) have been found in six patients. In addition, specific MAMLD1 cSNP(s) and haplotype may constitute a susceptibility factor for hypospadias. Furthermore, in vitro studies have revealed that (1) the mouse homolog is expressed in fetal Sertoli and Leydig cells around the critical period for sex development; (2) transient Mamld1 knockdown results in significantly reduced testosterone production primarily because of compromised 17α-hydroxylation and Cyp17a1 expression in Murine Leydig tumor cells; (3) MAMLD1 localizes to the nuclear bodies and transactivates the promoter activity of a non-canonical Notch target gene hairy/enhancer of split 3, without demonstrable DNA-binding capacity; and (4) MAMLD1 is regulated by steroidogenic factor 1 (SF1). These findings suggest that the MAMLD1 mutations cause 46,XY DSD primarily because of compromised testosterone production around the critical period for sex development. Further studies will provide useful information for the molecular network involved in fetal testosterone production. [ABSTRACT FROM AUTHOR]

The molecular steps in normal sexual development were largely discovered by studying patients and animal models with disorders of sexual development (DSD). Although several types of DSD have been reported in the cat and dog, which are often strikingly similar to human DSD, these have been infrequently utilized to contribute to our knowledge of mammalian sexual development. Canine and feline cases of DSD with sufficient evidence to be considered as potential models are summarized in this report. The consensus DSD terminology, and reference to previous terminology, is used to foster adoption of a common nomenclature that will facilitate communication and collaboration between veterinarians, physicians, and researchers. To efficiently utilize these unique resources as molecular tools continue to improve, it will be helpful to deposit samples from valuable cases into repositories where they are available to contribute to our understanding of sexual development, and thus improve human and animal health. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Dihydrotestosterone is crucial for normal development of external genitalia and prostate in the male embryo. Autosomal recessive mutations in the 5α-reductase type 2 (SRD5A2) gene disrupt the synthesis of dihydrotestosterone in the urogenital tract and give rise to genetic males with undervirilized external genitalia that may be female-like or ambiguous. In this study, three unrelated 46,XY children (0.5, 3, and 8 years old) who presented severe undermasculinization at birth were examined for genetic abnormalities in the SRD5A2 gene. Coding sequence abnormalities were ascertained by exon-specific polymerase chain reaction (PCR), single-stranded conformational polymorphism (SSCP), and sequencing analysis. Functional properties of the mutant alleles were investigated by means of site-directed mutagenesis assays. DNA molecular studies showed that all three patients were compound heterozygotes for SRD5A2 mutations. Patient 1 had a point mutation 547G → A in exon 3 (G183S) and a novel dinucleotidic mutation 634,635CC → TG in exon 4 (P212X). This double change results in premature termination signal (TGA) at codon 212, which predicts the expression of a truncated 211-amino acid protein. Patient 2 was the carrier of mutations G115D in exon 3 and S210F in exon 4. Patient 3 had two substitution mutations in exon 1, including a novel G → C transversion at nucleotide 169 (E57Q) and a G → A transition at nucleotide 254 (G85D). In transitory transfection assays, the recombinant cDNAs harboring mutations E57Q and G85D showed residual 5α-reductase activity, whereas those with mutations G115D, S210F, and P212X were devoided of activity. In contrast, the G183S substitution affected the catalytic activity of the enzyme by decreasing its affinity for testosterone substrate. We describe six different mutations of the SRD5A2 gene detected in three children with genital ambiguity. These genotypes are consistent with the clinical phenotype of steroid 5α-reductase 2 deficiency. Our data suggest that the combined gene variants (E57Q/G85D, G115D/S210F, and G183S/P212X) result in subfunctional or nonfunctional enzymes, causing masculinization defects in these patients. This further underscores that exon 4 of SRD5A2 may be a site prone to inactivating mutations. [ABSTRACT FROM AUTHOR]

Deficiency of 17β-hydroxysteroid dehydrogenase type3 (17β-HSD3) isoenzyme which catalyzes the synthesis of testosterone from Δ4-androstenedione, is the cause of 46, XY disorders of sex development (DSD). 17β-HSD3 deficiency is a rare autosomal recessive disorder, which is caused by mutations in the HSD17B gene found on chromosome 9q22. Up to now, almost 33 mutations in the HSD17B3 gene have been reported. Here, we report a patient with a novel mutation in HSD17B3 gene leading to 17β-HSD3 deficiency. The patient was admitted because of primary amenorrhea and signs of virilization at puberty. The chromosome analysis showed a 46, XY karyotype. Hormonal evaluation revealed a high Δ4-androstenedione level with a low serum testosterone/androstenedione (T/A) ratio. Sequence analysis of HSD17B3 gene revealed the presence of a homozygous missense mutation in exon 11 resulting in a premature stop codon (p.Y287). Gonadectomy was performed after the molecular diagnosis and estrogen replacement therapy was initiated. With this report, we emphasize that 17β-HSD3 deficiency should be considered in virilized female patients at puberty if the T/A ratio is less than 0.8, and the molecular analysis should be performed for the precise diagnosis and genetic counseling. [ABSTRACT FROM AUTHOR]

• A female patient with a classic picture of complete androgen insensitivity syndrome. • A novel mutation in the AR gene was identified by direct sequencing: p.R856L. • Functional studies were performed to investigate the functional properties of p.R856L. • p.R856L mutation strongly affected both transactivation capacity and N/C interaction. • Functional studies confirmed the pathogenicity of p.R856L mutation. Androgens are critical for male sex differentiation. Their actions are mediated by the androgen receptor (AR). Mutations disrupting AR function result in the androgen insensitivity syndrome (AIS). In this study, we identified in a patient with complete AIS, a novel AR mutation p.R856L. To investigate the functional properties of p.R856L, we performed functional studies. In comparison, we have characterized two already described mutations: p.R856H and p.R856C. We used a model composed of two different promoters fused to a reporter gene, two cell lines, and showed that all mutations were able to transactivate the (ARE) 2 -TATA promoter expressed in CHO cells more highly. Moreover, we confirmed the pathogenicity of the p.R856L and p.R856C mutations, and their associations with complete AIS. In contrast, the p.R856H mutation, which is associated with a spectrum of AIS phenotypes, showed less severe transcriptional constraints. Altogether, our studies allowed us to better characterize arginine residue at p.R856 position. [ABSTRACT FROM AUTHOR]

• Ten patients with a classic picture of complete androgen insensitivity syndrome. • Molecular analysis was established by direct sequencing of the AR gene. • Molecular diagnosis confirmed the presence of AR mutations in nine patients. • Two novel mutations in the AR gene were identified: IVS3 + 1G > T and p.288Qfs*14. • The novel mutations are predicted to be pathogenic and responsible for the phenotype. The Androgen insensitivity syndrome (AIS) in its complete form (CAIS) is a disorder in abnormal male development characterized by a complete female phenotype in a 46,XY individual. The most frequent cause of this disorder is a hemizygous mutation in androgen receptor (AR) gene located in X chromosome. The first aim of this study was to confirm the clinical diagnosis in a series of Tunisian patients with a typical phenotype of CAIS by molecular genetic analysis. The second aim was to determine the AR mutational profile in the local population. The entire coding region and the exon-intron junctions of the AR gene were sequenced in a series of ten patients. AR defects were found in nine patients. Despite the small number of cases, two of the nine identified mutations were novel. The first novel mutation was an 8-bp deletion in exon 1 (c.862_869del) resulting in a frameshift (p.A288Qfs*14). The second was a splice site mutation c.1885 + 1G > T (IVS3 + 1G > T). In this study, genetic testing has confirmed the diagnosis of most CAIS patients and has revealed two novel mechanisms responsible for the pathogenesis of AIS, as well as seven other reported mutations. [ABSTRACT FROM AUTHOR]